The cystic fibrosis transmembrane conductance regulator protein (CFTR) is a cAMP activated chloride ion (Cr) channel responsible for Cl− transport. CFTR is expressed in epithelial cells in mammalian airways, intestine, pancreas and testis. It is there where CFTR provides a pathway for the movement of Cl− ions across the apical membrane and a key point at which to regulate the rate of transepithelial salt and water transport. Hormones, such as a β-adrenergic agonist, or toxins, such as cholera toxin, lead to an increase in cAMP, activation of cAMP-dependent protein kinase, and phosphorylation of the CFTR Cl− channel, which causes the channel to open. An increase in the concentration of Ca2+ in a cell can also activate different apical membrane channels. Phosphorylation by protein kinase C can either open or shut Cl− channels in the apical membrane.
Dysfunction of CFTR is associated with a wide spectrum of disease, including cystic fibrosis (CF) and with some forms of male infertility, polycystic kidney disease and secretory diarrhea. CF is a hereditary disease that mainly affects the lungs and digestive system, causing progressive disability and early death. With an average life expectancy of around 31 years, CF is one of the most common life-shortening, childhood-onset inherited diseases. This disease is caused by mutation of the gene encoding CFTR, and is autosomal recessive. The most common CFTR mutation, deletion of phenylalanine-508 (ΔF508-CFTR), is present in at least one allele in about 90% of CF patients (Egan et al., (2004) Science 304:600-602). ΔF508-CFTR causes Cl− impermeability because it is not processed correctly, causing it to be retained at the endoplasmic reticulum (rather than the plasma membrane). ΔF508-CFTR also has reduced intrinsic Cl− conductance relative to wild type CFTR.
Strategies have been investigated to correct the defects in ΔF508-CFTR cellular processing and intrinsic function in cells. Cell growth at low temperature (<30° C.) (Denning et al., (1992) Nature 358, 761-764) or with high concentrations of chemical chaperones such as glycerol (Sato et al., (1996) J. Biol. Chem. 271, 635-638; Brown, et al., (1996) Cell Stress & Chaperones 1, 1 17-125) corrects partially defective ΔF508-CFTR cellular processing by a mechanism that may involve improved protein folding and stability (Sharma et al., (2001) J. Biol. Chem. 276, 8942-8950). A sustained increase in intracellular calcium concentration by thapsigargin also corrects defective ΔF508-CFTR processing (Egan et al., (2002) Nature Med. 8, 485-492), possibly by interfering with interactions with molecular chaperones. Compounds like phenylbutryate facilitate ΔF508-CFTR cellular processing by altering chaperone function and/or transcriptional enhancement (Rubenstein et al., (2000) Am. J. Physiol. 278, C259-C267; Kang et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 838-843). Although these approaches provide insight into mechanisms of ΔF508-CFTR retention at the endoplasmic reticulum, they probably do not offer clinically-useful therapies.
ΔF508-CFTR has significantly impaired channel activity even when present at the cell plasma membrane (Dalemans et al., (1991) Nature 354, 526-528). Cell-attached patchclamp measurements showed reduced ΔF508-CFTR open channel probability and prolonged closed times even with maximal cAMP stimulation (Haws et al., (1996) Am. J. Physiol. 270, C1544-C1555; Hwang et al., (1997) Am. J. Physiol. 273, C988-C998). Patch-clamp measurements in excised membranes indicated 7-fold reduced ΔF508-CFTR activation after phosphorylation compared to wildtype CFTR. Relatively high concentrations of the flavone genistein (>50 pM, Hwang, et al., (1997) Am. J. Physiol. 273, C988-C998; Wang et al., (2000) J. Physiol. 524, 637-638) or the xanthine isobutylmethylxanthine (>1 mM, Drurnrn et al., (1991) Science 254, 1797-1799) in combination with cAMP agonists increase ΔF508-CFTR channel activity. Again, these studies have not offered any clinically useful therapies.
Recent identification of small molecule bithiazole derivatives as correctors of mutant CFTR cellular processing or folding have been reported (WO 2006/101740). The derivatives were effective in the high nanomolar (nM) range. However, one of the most potent compounds reported in WO 2006/101740 has features that may impact optimal drug activity.
There is accordingly still a need for compounds that can correct cellular processing or folding of mutant CFTR, e.g., ΔF508-CTFR, and methods of using such compounds for the study and treatment of CF and the treatment and control of other secretory disorders. The present invention addresses these needs, as well as others.
Literature
Bithiazole derivatives as correctors of mutant CFTR cellular processing are reported in International Patent Application Publication (PCT) No. WO 2006/101470. Other PCT publications related to CFTR are represented by WO 01/55106 and WO 2005/120497.
Compounds, formulations and methods of diagnosing and treating mutant-CFTR associated disorders, and related literature of interest are reported in the following U.S. Pat. Nos. 3,953,428; 5,240,846; 5,366,977; 5,407,796; 5,434,086; 5,543,399; 5,582,825; 5,602,110; 5,621,007; 5,635,160; 5,639,661; 5,670,488; 5,674,898; 5,750,571; 5,776,677; 5,834,214; 5,840,702; 5,855,918; 5,863,770; 5,877,179; 5,908,611; 5,939,255; 5,939,536; 5,948,814; 5,958,893; 5,958,907; 5,972,995; 5,976,499; 5,981,178; 5,981,714; 5,989,521; 6,001,588; 6,015,828; 6,030,961; 6,033,688; 6,043,389; 6,063,913; 6,083,954; 6,093,567; 6,110,955; 6,130,248; 6,159,968; 6,201,107; 6,245,735; 6,251,930; 6,281,240; 6,323,187; 6,323,191; 6,329,422; 6,465,494; 6,573,073; 6,599,907; 6,630,482; 6,635,627; 6,723,703; 6,730,777; 6,770,739; 6,780,839; 6,902,907; 6,936,618; 6,984,487; 7,118,911; 7,160,729; 7,235,573; 7,238,474; 7,256,210; 7,258,854; 7,259,184; 7,259,250; 7,259,266; 7,261,102; 7,262,200; 7,264,926; 7,264,948; 7,265,088; 7,265,110; 7,265,114; 7,265,148; 7,265,153; 7,267,120; 7,267,652; 7,267,994; 7,268,134; 7,268,155; and 7,268,159.
Reports on the study and correction of defects in CFTR are found in the following references: Denning et al., (1992) Nature 358, 761-764; Sato et al., (1996) J. Biol. Chem. 271, 635-638; Brown, et al., (1996) Cell Stress & Chaperones 1, 1 17-125; Sharma et al., (2001) J. Biol. Chem. 276, 8942-8950; Egan et al., (2002) Nature Med. 8, 485-492; Rubenstein et al., (2000) Am. J. Physiol. 278, C259-C267; Kang et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 838-843; Dalemans et al., (1991) Nature 354, 526-528; Haws et al., (1996) Am. J. Physiol. 270, C1544-C1555; Hwang, et al., (1997) Am. J. Physiol. 273, C988-C998; Wang et al., (2000) J. Physiol. 524, 637-638; and Drurnrn et al., (1991) Science 254, 1797-1799.